Showing papers on "Alkaline phosphatase published in 2003"

Platelet‐Rich Plasma Contains High Levels of Platelet‐Derived Growth Factor and Transforming Growth Factor‐β and Modulates the Proliferation of Periodontally Related Cells In Vitro

Abstract: 849 Background: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. Methods: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-β1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. Results: In the PRP preparations, platelets were concentrated up to 70.9 × 10 4 cells/µl (283.4% of the unconcentrated plasma). The levels of PDGFAB and TGF-β1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. Conclusions: These data demonstrated that both PDGF-AB and TGF-β1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-β1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy. J Periodontol 2003;74:849-857.

Phosphatase activity in the sea

Abstract: Phosphatase is a key-enzyme in the marine environment, although life in the sea is normally not P limited. Expression of phosphatase in algae is generally regulated by the prevailing external concentration of inorganic phosphate, but the internal N:P ratio may also play a role. For bacteria, additional mechanisms like their C and N demands may be important. This is suggested by high phosphatase activities occasionally measured in eutrophic or deep water in the presence of relatively high phosphate concentrations. The distribution of phosphatase activity among the particulate and the dissolved fractions is highly variable. In particular, the dissolved fraction can contribute considerably to the total phosphatase activity (up to 70%), which differs from the pattern of other hydrolytic ectoenzymes. Parts of this fraction may originate from marine protozoa. The contribution of bacteria and phytoplankton to the particle-associated fraction of phosphatase is extremely variable, depending on P-availability, the dominant organisms, water depth and environmental factors. Community analysis revealed that bacteria attached to marine snow and N2-fixing cyanobacteria were frequently strong producers of phosphatase. Field studies carried out on a great variety of marine regions suggest that phosphatase activity is generally a good indicator of the P status of phytoplankton. Several heat-stable or heat-labile phosphatases, isolated from marine organisms living in extreme or other environments have been recommended for biotechnological applications.

Toxicity of cypermethrin in Labeo rohita fingerlings: biochemical, enzymatic and haematological consequences

Abstract: The effect of exposure to sub-lethal concentrations of cypermethrin, a synthetic pyrethroid pesticide, on biochemical parameters of muscle, blood and enzyme activities in brain, liver and kidney of the Indian major carp, Labeo rohita was studied. The sub-lethal exposure studies were done for up to 45 days at 1/10 and 1/50 of 96 h LC(50) of cypermethrin. The 96 h LC(50) was found to be 0.139 ppm. RNA levels decreased while DNA levels were elevated. Acid phosphatase was unchanged while alkaline phosphatase was depleted. Brain acetylcholinesterase activity was decreased significantly (P<0.05) over a period of 45 days at both cypermethrin concentrations. Lactate dehydrogenase activity in brain and liver was elevated, but inhibited in kidney. Succinate dehydrogenase and ATPase activities were depleted in brain, kidney and liver. There was a decrease in serum protein level over control at both concentrations of the pyrethroid. Blood glucose level and total leucocytes were elevated compared with controls at either concentration from day 15 to day 45. Haemoglobin percentage and total erythrocytes decreased in both sub-lethal concentrations. Extracts of the herb Datura stramonium were effective in countering the toxicity of this pesticide. Our data suggest that sub-lethal exposure of cypermethrin alters the biochemical, haematological parameters and enzymes of organs tissue and exert stress on the fish. Plant extracts may be useful in counteracting some of these effects.

Accelerated fat absorption in intestinal alkaline phosphatase knockout mice.

Abstract: Intestinal alkaline phosphatase (IAP) is the most ancestral of the tissue-specific members of the AP gene family. Several studies have suggested an absorptive function for IAP, but in vivo data to this effect have been lacking. We inactivated the mouse IAP gene in embryo-derived stem cells and generated mice homozygous for the null mutation. The mice were macroscopically and histologically normal and fertile and showed no difference from the wild-type controls under normal laboratory conditions. However, when maintained long-term on a high-fat diet, the IAP-deficient mice showed faster body weight gain than did control animals. Histological examination revealed an accelerated transport of fat droplets through the intestinal epithelium and elevation of serum triglyceride levels in the IAP-deficient mice compared to wild-type mice. Our study suggests that IAP participates in a rate-limiting step regulating fat absorption.

Calf Intestinal Alkaline Phosphatase, a Novel Therapeutic Drug for Lipopolysaccharide (LPS)-Mediated Diseases, Attenuates LPS Toxicity in Mice and Piglets

Abstract: It has been demonstrated that human placental alkaline phosphatase (HPLAP) attenuates the lipopolysaccharide (LPS)-mediated inflammatory response, likely through dephosphorylation of the lipid A moiety of LPS. In this study, it is demonstrated that also alkaline phosphatase derived from calf intestine (CIAP) is able to detoxify LPS. In mice administered CIAP, 80% of the animals survived a lethal Escherichia coli infection. In piglets, previous to LPS detoxification, the pharmacokinetic behavior of CIAP was studied. CIAP clearance was shown to be dose-independent and showed a biphasic pattern with an initial t1/2 of 3 to 5 min and a second phase t1/2 of 2 to 3 h. Although CIAP is cleared much faster than HPLAP, it attenuates LPS-mediated effects on hematology and tumor necrosis factor-alpha responses at doses up to 10 microg/kg in piglets. LPS-induced hematological changes were antagonized, and the tumor necrosis factor-alpha response was reduced up to 98%. Daily i.v. bolus administration of 4000 units CIAP, the highest dose used in the LPS intervention studies, in piglets for 28 days was tolerated without any sign of toxicity. Therefore, CIAP potentially encompasses a novel therapeutic agent in the treatment of LPS-mediated diseases. Based on the data mentioned above, human clinical trials have been initiated.

Alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis.

Abstract: The effect of alcohol on rabbit bone marrow and on the differentiation of mouse bone marrow stromal cells was investigated Alcohol was administered intragastrically at a dose of 10 mL/kg/day for 1 to 6 months Alcohol induced a significant increase in serum lipid peroxides, triglyceride, and cholesterol, and a reduction in superoxide dismutase activity Fatty infiltration in the liver and adipogenesis in bone marrow were found histologically after alcohol administration Fat cell hypertrophy and proliferation and diminished hematopoiesis in the subchondral area of the femoral head were observed Triglycerides were deposited in osteocytes, which became pyknotic, and the percentage of empty osteocyte lacunae increased None of these abnormal changes were detectable in the control group In the in vitro study, the marrow stromal cells were treated with increasing (003, 009, and 015 mol/L) concentrations of ethanol for 4 to 21 days Alcohol induced the differentiation of the cells into adipocytes The number of adipocytes increased with longer durations of exposure to ethanol and with higher concentrations Cells treated with ethanol also showed diminished alkaline phosphatase activity and expression of osteocalcin These novel findings indicate that alcohol can directly induce adipogenesis, decrease osteogenesis in bone marrow stroma, and produce intracellular lipid deposits resulting in the death of osteocytes, which may be associated with the development of osteonecrosis, especially in patients with long-term and excessive use of alcohol

The effect of hyaluronan on osteoblast proliferation and differentiation in rat calvarial‐derived cell cultures

Abstract: Hyaluronan (or hyaluronic acid, HA) is an essential component of extracellular matrices. It interacts with other macromolecules and plays a predominant role in tissue morphogenesis, cell migration, differentiation, and adhesion. The cell signaling functions of HA are mediated through the CD-44 receptor and are dependent upon the molecular weight of the polymer. We hypothesized that an HA of appropriate molecular weight alone in optimal concentration may induce osteoblast differentiation and bone formation. Enzyme-digested calvarial-derived mesenchymal cells from 2-day-old newborn rats were cultured with the addition of HA of three different molecular weights (2300, 900, and 60 kDa). We added, 0.5, 1.0, and 2.0 mg/mL HA for each molecular weight to the medium at the first plating of cells. After 7 to 20 days in culture, cell proliferation and differentiation were evaluated by measuring thymidine incorporation, alkaline phosphatase activity, and osteocalcin gene expression. The effects of HA on bone formation were examined by using Alizarin red staining for mineralization. The results showed that low molecular weight HA (60 kDa) significantly stimulated cell growth, increased osteocalcin mRNA expression in a dose-dependent manner, but showed no apparent effects on alkaline phosphatase activity and bone mineralization. On the other hand, high-weight HA (900 and 2,300 kDa) significantly increased all the parameters examined, particularly alkaline phosphatase activity, in a dose-dependent manner and stimulated cell mineralization to 126% and 119% of the controls, respectively, in the 1.0 mg/mL dose. Our findings suggest that HA has a molecular weight-specific and dose-specific mode of action that may enhance the osteogenic and osteoinductive properties of bone graft materials and substitutes due to its stimulatory effects on osteoblasts.

BMP responsiveness in human mesenchymal stem cells.

Abstract: Bone morphogenetic proteins (BMPs) are well known to induce bone formation in animal models and can promote osteogenesis in cultures of multipotential mesenchymal stem cells (MSC) isolated from rat and mouse bone marrow. However, clinical trials of BMPs suggest that BMPs are relatively ineffective inducers of osteogenesis in humans. Recent studies from our lab indicate that when human bone marrow MSC are placed in primary culture, osteogenesis can be induced by dexamethasone (Dex), but not by BMP-2, -4, or -7. We have therefore investigated components of BMP signaling pathways in human MSC. First passage cells, derived from the bone marrow of patients undergoing hip replacement surgery, were cultured with ascorbate phosphate and treated with 100 nM dexamethasone (Dex), 100 ng/ml BMP, or both. After 6 days, alkaline phosphatase activity of cell extracts was measured, and RNA was extracted for RT-PCR analysis of mRNA levels. Among human MSC samples from more than a dozen patients, only one patient sample showed significantly elevated alkaline phosphatase after exposure to BMP; the rest responded to Dex but not BMP. Analysis of mRNA from cultured human MSC indicated that, while Dex treatment caused increased levels of mRNA for alkaline phosphatase, BMP did not. Noggin is a BMP-binding protein that is upregulated by BMPs. BMP-treated human MSC cultures that did not show increased alkaline phosphatase did express elevated levels of noggin mRNA, indicating that the cells are capable of some BMP response. Our results suggest that BMP signaling in mesenchymal stem cells utilizes more than one system for transcriptional activation. The inability of most human MSC to activate transcription of the alkaline phosphatase gene implies that a defect exists in the system required for induction of the osteoblast phenotype.

Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells

Abstract: Study Design. In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells. Objectives. To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteogiycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells. of Background Data. BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondtogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteogiycan synthesis in articular chondrocytes and IVD cells. Nevestlieless. the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histocnemical stains. Materials and Methods. Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bore of illum for histochemical stains. Recombinant human BMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells alter transduction of BMP-2 cDNA, then concentratec and purified. Then IVD cell cuitures were exposed to various concentrations of thBMP-2. Reverse transcription-polymerse chain reaction for mRNA expression of aggretacan, collagen type 1, collagen type II, and osteacaicin was performed. Newly synthesized pruieoglycan was measured by 35S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As 5 histochemical examination, alkaline phosphatase and Alitarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectivety. Resuws. in the rhBMP-2 treated cultures, there was increased newly synthesized prateoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL. of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type Ii mRNA over untreated. control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Aiizarin red-S, while osteoblast curture exhibited strong positive staint. Conclusions. The rhBMP-2 clearty up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenie phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.

Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice.

Abstract: The present study investigated the prophylactic influence of melatonin against cyclophosphamide-induced oxidative stress in mouse tissues. Lipid peroxidation, reduced glutathione (GSH), glutathione disulphide (GSSG), glutathione peroxidase (GSH-Px) and serum phosphatase levels were analyzed in brain, spleen liver, lungs, kidney and testes. Fifteen days oral administration with melatonin (0.1 mg/kg bw per day) before treatment checked the augmentation of the level of lipid peroxidation, blood GSSG and acid phosphatase caused by an acute treatment with a radiomimetic drug, cyclophosphamide (75 mg/kg bw). Cyclophosphamide-induced depletion in the level of GSH, GSH-Px and alkaline phosphatase was made up statistically significant by chronic melatonin administration given orally. The results indicate the antioxidative properties of melatonin resulting into its prophylactic property against the cyclophosphamide-induced biochemical alterations. The finding support the idea that melatonin is a potent free-radical scavenger and antioxidant.

Regulation of BMP-induced transcription in cultured human bone marrow stromal cells.

Abstract: Background: Adherent bone marrow stromal cells are inducible osteoprogenitors, giving rise to cells expressing osteoblast markers including alkaline phosphatase, osteopontin, osteocalcin, and bone sialoprotein. However, the potency of inducers varies in a species-specific manner. Glucocorticoids such as dexamethasone induce alkaline phosphatase activity in both human and rat mesenchymal stem cells, while mouse bone marrow stromal cells are refractory to dexamethasone-induced alkaline phosphatase activity. In contrast, BMP induces alkaline phosphatase activity in both mouse and rat bone marrow stromal cells, while BMP effects on human bone marrow stromal cells are poorly characterized. Methods: Bone marrow samples were isolated from patients undergoing hip replacement. Mononuclear marrow cells were cultured and grown to confluence without or with 10 -7 M dexamethasone. Cells from each isolate were passaged into medium containing 100 μg/mL ascorbate phosphate and treated with dexamethasone, 100 ng/mL BMP, or no inducer. At day 6, alkaline phosphatase activity was assayed, and RNA was prepared for mRNA analyses by real-time polymerase chain reaction. Results: Bone marrow stromal cells from twenty-four of twenty-six patients showed no significant osteogenic response to BMP-2, 4, or 7 as determined by alkaline phosphatase induction. However, BMPs induced elevated levels of other genes associated with osteogenesis such as bone sialoprotein and osteopontin as well as BMP-2 and noggin. If primary cultures of human bone marrow stromal cells were pretreated with dexamethasone, BMP-2 treatment of first-passage cells induced alkaline phosphatase in approximately half of the isolates, and significantly greater induction was seen in cells from males. Dexamethasone treatment, like BMP treatment, also increased expression of the BMP-binding protein noggin. Conclusions: Most human femur bone marrow stromal cell samples appear incapable of expressing elevated alkaline phosphatase levels in response to BMPs. Since BMP treatment induced expression of several other BMP-regulated genes, the defect in alkaline phosphatase induction is presumably not due to impaired BMP signaling. We hypothesize that the mechanism by which BMPs modulate alkaline phosphatase expression is indirect, involving a BMP-regulated transcription factor for alkaline phosphatase expression that is controlled differently in humans and rodents. Clinical Relevance: We suggest that the relative insensitivity of alkaline phosphatase to BMP induction in human bone marrow stromal cells may contribute to the variation in efficacy reported with BMP in clinical settings.

Type I collagen production by osteoblast-like cells cultured in contact with different bioactive glasses.

Abstract: Bioactive glasses are silica-based, surface-active bone substitutes that have shown good biocompatibility both in bone and in soft tissue and are used in oral and maxillofacial bone augmentation. Previous in vitro studies showing that bioactive glasses support the growth and maturation of rat osteoblast-like cells and promote the expression and maintenance of the osteoblastic phenotype have suggested that there is both a solution-mediated and a surface-controlled effect on cell activity. In this study, we investigated the behavior of human primary osteoblast-like cells cultured in contact with three different bioactive glasses and compared them with amorphous silica (SiO2) used in the form of granules. The specific activity of alkaline phosphatase determined biochemically was significantly higher at 2 and 4 days on the bioactive glass with 46.1 mol % silica content (45S5 Bioglass) cultures than in the control cultures and in the bioactive gel-glass cultures, which had 60 mol % (58S) and 80 mol % (77S) silica content. Osteoblasts synthesize collagen type I, which is subsequently mineralized. Immunoblot and biochemical studies showed increased collagen release from osteoblast-like cells cultured in contact with bioactive glasses over that of controls. Among the three bioactive glasses, 45S5 is the highest inducer of osteoblast-like cell collagen release; moreover, mRNA for type I collagen was stimulated approximately three- to fivefold after 45S5 treatment. 77S bioactive glass similarly increased type I collagen synthesis even though alkaline phosphatase was not higher. These results suggest that 45S5 Bioglass not only induces osteogenic differentiation of human primary osteoblast-like cells, but can also increase collagen synthesis and release. The newly formulated bioactive gel-glass 77S seems to have potential applications for tissue engineering, inducing increased collagen synthesis.

Additive effects of copper and zinc on cadmium toxicity on phosphatase activities and ATP content of soil as estimated by the ecological dose (ED50)

Abstract: The ecological dose (ED50) of Cd on alkaline and acid phosphatase activity and the ATP content of three contrasting forest soils was measured with or without Cu and Zn to assess the additive toxic effects of these two metals. Soils polluted with Cu and/or Zn were treated with increasing Cd concentrations to give the following metal combinations: Cd, Cd+Cu, Cd+Zn and Cd+Cu+Zn. Alkaline and acid phosphatase activities and ATP content of the three soils were analysed 4 h, 7 and 28 days after the metal additions. The ED50 values were obtained by interpolating the enzyme activities or ATP data with a kinetic model and the goodness of fit was satisfactory. Generally, the ED50 values of both acid and alkaline phosphatase activities for Cd were lower (higher toxicity) with than without Cu and Zn and the effect of Cu and Zn was particularly adverse when these two metals were both added to soils. The alkaline phosphatase was more sensitive in the acid and neutral soil whereas the acid phosphatase was more sensitive in the alkaline soil. Both phosphatase activities and the ATP content were more sensitive in the sandy than in the finer textured soils. The ATP content was less sensitive to the additive effects. Increasing toxicity was observed during the incubation. Analysis of 1 M NH4NO3-extractable Cd, Cu and Zn revealed that Cd competed with Zn for the adsorption sites but not with Cu. However, the lower ED50 values for Cd of the two phosphatase activities and of the ATP content in the presence of heavy metal combinations could be not explained by the heavy metal solubility data. It is concluded that the ED50 may be a sensitive tool for assessing additve toxic effects to soil biochemical parameters.

Pectin and pectic-oligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma cells.

Abstract: Background Dietary fibres have been associated with decreased risk of various cancers, although the mechanisms are unclear. Induction of apoptosis in tumour cells is thought to be an important protective mechanism against colorectal cancer. This work investigates the effects of pectins and pectic-oligosaccharides (POS) on the human colonic adenocarcinoma cell line HT29. Materials and methods The anti-proliferative effects of pectin and POS were studied by testing the HT29 cells for cytotoxicity, differentiation and/or apoptosis by lactate dehydrogenase, alkaline phosphatase and caspase-3 activity assays. DNA agarose gel electrophoresis was also carried out. Results A significant reduction in attached cell numbers was observed after three days incubation. This decrease was neither due to cells undergoing necrosis nor differentiation. Increased apoptosis frequency, after incubation with 1% (w/v) pectin and/or POS, was demonstrated by caspase-3 activity and DNA laddering on agarose gel electrophoresis. Conclusion Dietary pectins and their degradation products may contribute to the reported protective effects of fruits against colon cancer.

Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts.

Abstract: Background: Periodontal ligament fibroblasts (PDLFs) are the cells essential for periodontal regeneration. PDLFs comprise a heterogeneous cell population and consist of several cell subsets that differ in their function. It is known that PDLFs produce osteoblast-related extracellular matrix proteins and show higher alkaline phosphatase (ALP) activity compared with gingival fibroblasts (GFs), implying that PDLFs have osteogenic characterisitics. The aim of the present study was to isolate the osteogenic population of PDLFs according to their expression of ALP. Methods: PDLFs and gingival fibroblasts were separated into two populations, ALP-positive and ALP-negative, with an immunomagnetic method using a monoclonal antibody against human bone type ALP and magnetic beads conjugated with a secondary antibody. Expression of basic fibroblast growth factor (bFGF) receptor and transforming growth factor (TGF)-β receptor was investigated in these two populations. Osteoblastrelated molecules, osteocalcin, and bone ...

Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase

Abstract: Background Ursolic acid (UA) is a plant component with anti-inflammatory and anti-proliferative properties. Its effect on colon cancer is not clear. We studied the anticancer effects of UA on human colon cancer cells. Materials and methods HT-29 cells were treated with UA. Cell proliferation was determined by cleavage of WST-1. Apoptosis was assayed by cytosolic DNA-histone complex and caspase activity. Sphingomyelinase and alkaline phosphatase were determined against specific substrates. Results UA dose-dependently decreased cell proliferation and induced apoptosis, accompanied by activation of caspase 3, 8 and 9. Its antiproliferative effect was stronger than those of sulindac and camptothecin and its apoptotic effect stronger than those of boswellic acid and sulindac. UA selectively increased the activity of intestinal alkaline sphingomyelinase, which occurred before activation of caspases. UA had no effect on alkaline phosphatase activity. Conclusion UA has strong anti-proliferative and apoptotic effects on HT-29 cells. The effects may be mediated by alkaline sphingomyelinase activation.

Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures.

Abstract: The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 μM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids. Introduction: Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts. Materials and Methods: MC3T3-E1 cells were treated with dexamethasone (DEX; 1 μM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Results: DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for α1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for α1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX. Conclusions: Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of GCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.

The role of annexin 2 in osteoblastic mineralization.

Abstract: While the basic cellular contributions to bone differentiation and mineralization are widely accepted, the regulation of these processes at the intracellular level remains inadequately understood. Our laboratory recently identified annexin 2 as a protein involved in osteoblastic mineralization. Annexin 2 was overexpressed twofold in SaOSLM2 osteoblastic cells as a fusion protein with green fluorescent protein. The overexpression of annexin 2 led to an increase in alkaline phosphatase activity as well as an increase in mineralization. Our data suggest that the increase in alkaline phosphatase activity does not result from increased alkaline phosphatase transcript or protein levels; therefore we evaluated mechanism of action. We determined that both annexin 2 and alkaline phosphatase activity were localized to membrane microdomains called lipid rafts in osteoblastic cells. Annexin 2 overexpression resulted in an increase in alkaline phosphatase activity that was associated with lipid microdomains in a cholesterol-dependent manner. Furthermore, disruption of lipid rafts with a cholesterol sequestering agent or reduction of annexin 2 expression by specific antisense oligonucleotides each resulted in diminished mineralization. Therefore, intact lipid rafts containing annexin 2 appear to be important for alkaline phosphatase activity and may facilitate the osteoblastic mineralization process.